Development and Validation of a Sensitive Enzymeimmunoassay for Determination of Plasma Metastin in Mithun (Bos frontalis)

Metastin, also known as kisspeptin-10, is a potent stimulator of gonadotropin-releasing hormone (GnRH) neurons in the central nervous system. Recently, it has been emerged as a key player in the regulation of reproduction in mammals. Blood concentrations of metastin during different physiological stages in bovine species in general and mithun (Bos frontalis) in particular are not available. Lacking of such information may probably be due to non-availability of simple assay procedure to measure the peptide. Therefore, the objective of this study was to develop and validate a simple and sufficiently sensitive enzyme immunoassay (EIA) for metastin determination in mithun plasma using the biotin-streptavidin amplification system and second antibody coating technique. Biotin was coupled to metastin and used to bridge between streptavidin-peroxidase and the immobilized metastin antiserum in the competitive assay. The EIA was conducted directly in 150 μL of unknown mithun plasma. Metastin standards ranging from 0.01–51.2 ng/150 μL/well were prepared in hormone-free plasma. The lowest detection limit was 0.07 ng/mL plasma. Plasma volumes for the EIA, viz., 75, 150, and 200 μL did not influence the shape of standard curve even though a drop in OD₄₅₀ was seen with higher plasma volumes. A parallelism test was carried out to compare the endogenous mithun metastin with metastin standard used. It showed good parallelism with the metastin standard curve. For the biological validation of the assay, metastin was measured in (a) blood samples collected from 12 pregnant mithun cows during different stages of pregnancy, (b) in blood from seven early pregnant and 12 non-pregnant mithuns, and (c) in follicular fluid obtained from different types of follicle. It was found that the plasma metastin concentrations increased (P < 0.001) from first through last trimester of pregnancy. Plasma metastin levels were much higher (P < 0.001) in early pregnant than non-pregnant cows. Follicular fluid metastin concentrations were found to increase (P < 0.001) as the follicles grow and the highest levels were recorded in preovulatory follicles. In conclusion, a simple, sufficiently sensitive and direct EIA procedure has been developed for the first time to determine metastin levels in mithun. A wide range of metastin concentrations can be detected during different physiological stages in mithun using this metastin-EIA procedure.     

大额牛与德宏高峰牛种间杂交的亲子鉴定初报

利用7个微卫星座位对大额牛(Bos frontalis)与德宏高峰牛(Bos taurus)种间杂交后代进行微卫星座位分型,结果7个微卫星位点在亲子关系中遵循经典的孟德尔遗传规律,呈共显性遗传,证实了大额牛与德宏高峰牛种间杂交的可行性.     

Development and Application of a Sensitive,Second Antibody Format Enzymeimmunoassay (EIA) for Estimation of Plasma FSH in Mithun (Bos frontalis)

Mithun (Bos frontalis) is a semi-wild rare ruminant species. A simple sensitive enzymeimmunoassay suitable for assaying FSH in the blood plasma of mithun is not available which thereby limits our ability to understand this species reproductive processes. Therefore, the aim of this article was to develop a simple and sensitive enzymeimmunoassay (EIA) for estimation of FSH in mithun plasma and apply the assay to understand the estrous cycle and superovulatory process in this species. To accomplish this goal, biotinylated FSH was bridged between streptavidin-peroxidase and immobilized antiserum in a competitive assay. Forty microlitre mithun plasma was used directly in the EIA. The FSH standards were prepared in hormone free plasma and ranged from 5–1280 pg/well/40 μL. The sensitivity of EIA was 5 pg/well FSH, which corresponds to 0.125 ng/mL plasma and the 50% relative binding sensitivity was 90 pg/well/40 μL. Although the shape of the standard curve was not influenced by different plasma volumes viz. 40 and 80 μL, a slight drop in the OD₄₅₀ was observed with the increasing volume of plasma. Parallelism tests conducted between the endogenous mithun FSH and bovine FSH standards showed good homology between them. Plasma FSH estimated using the developed EIA and commercially available FSH EIA kit in the same samples were correlated (r = 0.98) and showed linearity. Both the Intra- and inter-assay CV were below 6%. Recovery of known concentrations of added FSH showed linearity (r = 0.99). The developed EIA was further validated biologically by estimating FSH in cyclic cows for the entire estrous cycle, in mithun heifers administered with GnRH analogues and in mithun cows during superovulatory treatment with FSH. In conclusion, the EIA developed for FSH determination in mithun blood plasma is simple and highly sensitive for estimation of mithun FSH in all physiological conditions.     

Isolation,identification and retrospective study of foot‐and‐mouth disease virus from affected Mithun (Bos frontalis) in north‐eastern India

Foot‐and‐mouth disease (FMD ) is a contagious disease of cloven‐hoofed animals that causes substantial and perpetual economic loss. Apart from the contagious nature of the disease, the FMD virus can establish in a “carrier state” among all cloven‐hoofed animals. The Mithun (Bos frontalis ), popularly called the “Cattle of Mountain,” is found in the geographically isolated, hilly region of north‐east India: Arunachal Pradesh, Nagaland, Manipur and Mizoram. Despite the geographical inaccessibility, infection by FMD virus has emerged as the single most devastating disease among Mithun after the eradication of rinderpest from this region. Samples from outbreaks of FMD in Mithun were analysed by sandwich ELISA , multiplex RT ‐PCR (MRT ‐PCR ) and liquid‐phase blocking enzyme‐linked immunosorbent assay and isolated in the BHK ‐21 cell line. The results indicate the presence of FMDV serotype “O.” The sequencing and molecular phylogenies have revealed close relationships in the lineage of type “O” isolates from Bangladesh. The findings will provide useful information for further research and development of a sustainable programme for the progressive control of FMD in the Mithun population.